The present invention relates to methods of generating infectious recombinant negative-strand RNA virses in mammalian cells from expression vectors in the absence of helper virus. The present invention also relates to methods of generating infectious recombinant negative-strand RNA viruses which have mutations in viral genes and/or which express, package and/or present peptides or polypeptides encoded by heterologous nucleic acid sequences. The present invention further relates the use of the recombinant negative-strand RNA viruses or chimeric negative-strand RNA viruses of the invention in vaccine formulations and pharmaceutical compositions.
A number of DNA viruses have been genetically engineered to direct the expression of heterologous proteins in host cell systems (e.g., vaccinia virus, baculovirtis, etc.). Recently, similar advances have been made with positive-strand RNA viruses (e.g., poliovirus). The expression products of these constructs, i.e., the heterologous gene product or the chimeric virus which expresses the heterologous gene product, are thought to be potentially useful in vaccine formulation is (either subunit or whole virus vaccines). One drawback to the use of viruses such as vaccinia for constructing recombinant or chimeric viruses for use in vaccines is the lack of variation in its major epitopes. This lack of variability in the viral strains places strict limitations on the repeated use of chimeric vaccinia, in that multiple vaccinations will generate host-resistance to the strain so that the inoculated virus cannot infect the host. Inoculation of a resistant individual with chimeric vaccinia will, therefore, not induce immune stimulation.
By contrast, the negative-strand RNA viruses, would be attractive candidates for constructing chimeric viruses for use in vaccines. The negative-strand RNA virus, influenza, for example is desirable because its wide genetic variability allows for the construction of a vast repertoire of vaccine formulations which stimulate immunity without risk of developing a tolerance.
The RNA-directed RNA polynerases of animal viruses have been extensively studied with regard to many aspects of protein structure and reaction conditions. However, the elements of the template RNA which promote optimal expression by the polynerase could only be studied by inference using existing viral RNA sequences. This promoter analysis is of interest since it is unknown how a viral polymerase recognizes specific viral RNAs from among the many host-encoded RNAs found in an infected cell.
Animal viruses containing plus-sense genome RNA can be replicated when plasmid-derived RNA is introduced into cells by transfection (for example, Racaniello et al., 1981, Science 214:916-919; and Levis, et al., 1986, Cell 44:137-145). In the case of poliovirus, the purified polymerase will replicate a genome RNA in in vitro reactions and when this preparation is transfected into cells it is infectious (Kaplan, et al., 1985, Proc. Natl. Acad. Sci. USA 82:8424-8428). However, the template elements which serve as transcription promoter for the poliovirus-encoded polymerase are unknown since even RNA homopolymers can be copied (Ward, et al., 1988, J. Virol. 62:558-562). SP6 transcripts have also been used to produce model defective interfering (DI) RNAs for the Sindbis viral genome. When the RNA is introduced into infected cells, it is replicated and packaged. The RNA sequences which were responsible for both recognition by the Sindbis viral polymerase and packaging of the genome into virus particles were shown to be within 162 nucleotides (nt) of the 5xe2x80x2 terminus and 19 nt of the 3xe2x80x2 terminus of the genome (Levis, et al., 1986, Cell 44:137-145). In the case of brome mosaic virus (BMV), a positive strand RNA plant virus, SP6 transcripts have been used to identify the promoter as a 134 nt tRNA-like 3xe2x80x2 terminus (Dreher, and Hall, 1988, J. Mol. Biol. 201:31-40). Polymerase recognition and synthesis were shown to be dependent on both sequence and secondary structural features (Dreher, et al., 1984, Nature 311:171-175).
The negative-sense RNA viruses have been refractory to study of the sequence requirements of the replicase. The purified polymerase of vesicular stomatitis virus is only active in transcription when virus-derived ribonucleoprotein complexes (RNPs) are included as template (De and Banerjee, 1985, Biochem. Biophys. Res. Commun. 126:40-49; Emerson and Yu, 1975, J. Virol. 15:1348-1356; Naito, and Ishihama, 1976, J. Biol. Chem. 251:4307-4314). With regard to influenza viruses, it was reported that naked RNA purified from virus was used to reconstitute RNPs. The viral nucleocapsid and polymerase proteins were gel-purified and renatured on the viral RNA using thioredoxin (Szewczyk, et al., 1988, Proc. Natl. Acad. Sci. USA 85:7907-7911). However, these authors did not show that the activity of the preparation was specific for influenza viral RNA, nor did they analyze the signals which promote transcription.
Only recently has it been possible to recover negative strand RNA viruses using recombinant reverse genetic techniques (see, e.g., U.S. Pat. No. 5,166,087, which is incorporated herein by reference in its entirety). In one embodiment of the reverse genetic technique, ribonucleoprotein complexes (RNPs) are reconstituted in vitro from RNA transcribed from plasmid DNA in the presence of influenza virus polymerase proteins (PB1, PB2 and PA) and nucleoprotein (NP) isolated from purified influenza virus (Enami et al., 1990, Proc. Natl. Acad. Sci. USA 87:3802-3805; Enami and Palese, 1991, J. Virol. 65:2711-2713; and Muster and Garcia-Sastre, Genetic manipulation of influenza viruses in Textbook of influenza (1998), ch. 9, eds. Nicholson et al.). The in vitro reconstituted RNPs are transfected into cells infected with a helper influenza virus, which provides the remaining required viral proteins and RNA segments to generate transfectant viruses. In another embodiment of the reverse genetic technique, RNPs are reconstituted intracellularly from plasmids expressing influenza virus polymerase proteins, nucleoprotein, and an influeniza-like vRNA segment (Neumann et al., 1994, Virology 202:477-479; Zhang et al., 1994, Biochem. Biophys. Res. Comm. 200:95-101; and Pleschka et al., J. Virol., 1996, 70:41 88-4192). The RNPs are packaged into transfectant viruses upon infection with helper influenza virus.
Virus families containing enveloped single-stranded RNA of the negative-sense genome are classified into groups having non-segmented genomes (Paramyxoviridae, Rhabdoviridae, Filoviridae and Borna Disease Virus) or those having segmented genomes (Orthomyxoviridae, Bunlyaviridae and Arenaviridae). The Orthomyxoviridae family, described in detail below, and used in the examples herein, includes the viruses of influenza, types A, B and C viruses, as well as Thogoto and Dhori viruses and infectious salmon anemia virus.
The influenza virions consist of an internal ribonucleoprotein core.(a helical nucleocapsid) containing the single-stranded RNA genome, and an outer lipoprotein envelope lined inside by a matrix protein (M1). The segmented genome of influenza A virus consists of eight molecules (seven for influenza C) of linear, negative polarity, single-stranded RNAs which encode ten polypeptides, including: the RNA-dependent RNA polymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which form the nucleocapsid; the matrix membrane proteins (M1, M2); two surface glycoproteins which project from the lipid containing envelope: hemagglutinin (HA) and neuraminidase (NA); the nonstructural protein (NS1) and nuclear export protein (NEP). Transcription and replication of the genome takes place in the nucleus and assembly occurs via budding on the plasma membrane. The viruses can reassort genes during mixed infections.
Influenza virus adsorbs via HA to sialyloligosaccharides in cell membrane glycoproteins and glycolipids. Following endocytosis of the virion, a conformational change in the HA molecule occurs within the cellular endosome which facilitates membrane fusion, thus triggering uncoating. The nucleocapsid migrates to the nucleus where viral mRNA is transcribed. Viral mRNA is transcribed by a unique mechanism in which viral endonuclease cleaves the capped 5xe2x80x2-terminus from cellular heterologous mRNAs which then serve as primers for transcription of viral RNA templates by the viral transcriptase. Transcripts terminate at sites 15 to 22 bases from the ends of their templates, where oligo(U) sequences act as signals for the addition of poly(A) tracts. Of the eight viral RNA molecules so produced, six are monocistronic messages that are translated directly into the proteins representing HA, NA, NP and the viral polynerase proteins, PB2, PB1 and PA. The other two transcripts undergo splicing, each yielding two mRNAs which are translated in different reading frames to produce M1, M2, NS1 and NEP. In other words, the eight viral RNA segments code for ten proteins: nine structural and one nonstructural. A summary of the genes of the influenza virus and their protein products is shown in Table 1 below.
The influenza A virus genome contains eight segments of single-stranded RNA of negative polarity, coding for one nonstructural and nine structural proteins. The nonstructural protein NS1 is abundant in influenza virus infected cells, but has not been detected in virions. NS1 is a phosphoprotein found in the nucleus early during infection and also in the cytoplasm at later times of the viral cycle (King et al., 1975, Virology 64:378). Studies with temperature-sensitive (ts) influenza mutants carrying lesions in the NS gene suggested that the NS1 protein is a transcriptional and post-transcriptional regulator of mechanisms by which the virus is able to inhibit host cell gene expression and to stimulate viral protein synthesis. Like many other proteins that regulate post-transcriptional processes, the NS1 protein interacts with specific RNA sequences and structures. The NS1 protein has been reported to bind to different RNA species including: vRNA, poly-A, U6 snRNA, 5xe2x80x2 untranslated region as of viral mRNAs and ds RNA (Qiu et al., 1995, RNA 1:304; Qiu et al., 1994, J. Virol. 68:425; and Hatada Fukuda 1992, J. Gen. Virol. 73:3325-9). Expression of the NS1 protein from cDNA in transfected cells has been associated with several effects: inhibition of nucleo-cytoplasmic transport of mRNA, inhibition of pre-mRNA splicing, inhibition of host mRNA polyadenylation and stimulation of translation of viral mRNA (Fortes et al., 1994, EMBO J. 13: 704; Enami et al, 1994, J. Virol. 68:1432; de la Luna et al., 1995, J. Virol. 69:2427; Lu et al., 1994, Genes Dev. 8:1817; Park et al., 1995, J. Biol Chem. 270:28433; Nemerof et al., 1998, Mol. Cell. 1:991; and Chen et al., 1994, EMBO J. 18:2273-83).
Influenza remains a constant xworl-dwide threat to human health and hence there is a particular need for a ready method of generating modified influenza viruses with known mutations in any of the genomic viral RNA (vRNA) segments. Engineering influenza vRNA segments for expression of heterologous sequences is also of much interest, for example, in the development of new vaccines effective against influenza virus and a second pathogenic agent.
The Paramyxoviridae family, described in detail below, and used in the examples herein, contain the viruses of Newcastle disease virus (NDV), parainfluenza virus, Sendai virus, simian virus 5, and mumps virus. The Newcastle disease virus is an enveloped virus containing a linear, single-strand, nonsegmented, negative sense RNA genome. The genoinic RNA contains genes in the order of 3xe2x80x2-NP-P-M-F-HN-L, described in further detail below. The genomic RNA also contains a leader sequence at the 3xe2x80x2 end.
The structural elements of the virion include the virus envelope which is a lipid bilayer derived from the cell plasma membrane. The glycoprotein, hemagglutinin-neuraminidase (HN) protrude from the envelope allowing the virus to contain both hemagglutinin and neuraminidase activities. The fusion glycoprotein (F), which also interacts with the viral membrane, is first produced as an inactive precursor, then cleaved post-translationally to produce two disulfide linked polypeptides. The active F protein is involved in penetration of NDV into host cells by facilitating fusion of the viral envelope with the host cell plasma membrane. The matrix protein (M), is involved with viral assembly, and interacts with both the viral membrane as well as the nucleocapsid proteins.
The main protein subunit of the nucleocapsid is the nucleocapsid protein (NP) which confers helical symmetry on the capsid. In association with the nucleocapsid are the P and L proteins. The phosphoprotein (P), which is subject to phosphorylation, is thought to play a regulatory role in transcription, and may also be involved in methylation, phosphorylation and polyadenylation. The L gene, which encodes an RNA-dependent RNA polymerase, is required for viral RNA synthesis together with the P protein. The L protein, which takes up nearly half of the coding capacity of the viral genome is the largest of the viral proteins, and plays an important role in both transcription and replication.
The replication of all negative-strand RNA viruses, including NDV, is complicated by the absence of cellular machinery required to replicate RNA. Additionally, the negative-strand genome can not be translated directly into protein, but must first be transcribed into a positive-strand (mRNA) copy. Therefore, upon entry into a host cell, the virus can not synthesize the required RNA-dependent RNA polymerase. The L, P and NP proteins must enter the cell along with the genome on infection.
It is hypothesized that most or all of the viral proteins that transcribe NDV mRNA also carry out their replication. The mechanism that regulates the alternative uses (i.e., transcription or replication) of the same complement of proteins has not been clearly identified but appears to involve the abundance of free forms of one or more of the nucleocapsid proteins, in particular, the NP. Directly following penetration of the virus, transcription is initiated by the L protein using the negative-sense RNA in the nucleocapsid as a template. Viral RNA synthesis is regulated such that it produces monocistronic mRNAs during transcription.
Following transcription, virus oeme replication is the second essential event in infection by negative-strand RNA viruses. As with other negative-strand RNA viruses, virus genome replication in Newcastle disease virus (NDV) is mediated by virus-specified proteins. The first products of replicative RNA synthesis are complementary copies (i.e., plus-polarity) of NDV genome RNA (cRNA). These plus-stranded copies (anti-genomes) differ from the plus-strand mRNA transcripts in the structure of their termini. Unlike the mRNA transcripts, the anti-genomic cRNAs are not capped and methylated at the 5xe2x80x2 termini, and are not truncated and polyadenylated at the 3xe2x80x2 termini. The cRNAs are coterminal with their negative strand templates and contain all the genetic information in each genomic RNA segment in the complementary form. The cRNAs serve as templates for the synthesis of NDV negative-strand viral genomes (vRNAs).
Both the NDV negative strand genomes (vRNAs) and antigenomes (cRNAs) are encapsidated by nucleocapsid proteins; the only unencapsidated RNA species are virus mRNAs. For NDV, the cytoplasm is the site of virus RNA replication, just as it is the site for transcription. Assembly of the viral components appears to take place at the host cell plasma membrane and mature virus is released by budding.
The present invention provides methods of generating infectious recombinant negative-strand RNA viruses intracellularly in the absence of helper virus from expression vectors comprising cDNAs encoding the viral proteins necessary to form ribonucleoprotein complexes (RNPs) and expression vectors comprising cDNA for genomic viral RNA(s) (vRNAs) or the corresponding cRNA(s). In particular, the present invention provides methods of generating infectious recombinant negative-strand RNA viruses in 293T cells in the absence of helper virus from expression vectors comprising cDNAs encoding the viral proteins necessary to form RNPs and expression vectors comprising cDNA for vRNA(s) or the corresponding cRNA(s). The infectious recombinant negative-strand RNA viruses of the invention may or may not be capable of replicating and producing progeny. The present invention encompasses methods of generating infectious recombinant negative-strand RNA viruscs having segmented or non-segmented genomes.
In one embodiment, an infectious recombinant negative-strand RNA virus having a segmented or non-segmented genome is rescued in a method comprising introducing into a 293T cell expression vectors capable of expressing the genomic or antigenomic RNA segments, and a nucleoprotein, and a RNA-dependent polymerase, whereby ribonucleoprotein complexes are formed and the recombinant negative-strand RNA virus is produced in the absence of helper virus. In accordance with this embodiment, the expression of the genomic vRNA(s) or the corresponding cRNA(s) and/or the expression of the nucleoprotein and RNA-dependent RNA polymerase may be constitutive or inducible. For example, the expression of the vRNA(s) or cRNA(s) under the control of a DNA-dependent RNA polymerase promoter such as the bacteriophage T7 promoter may be induced by inducing the expression of a DNA-dependent RNA polymerase such as T7.
In another embodiment, an infectious recombinant negative-strand RNA virus having a segmented or non-segmented genome is generated in 293T cells by a method comprising: (a) introducing expression vectors capable of expressing in said cells genomic vRNA(s) or the corresponding cRNA(s); (b) introducing expression vectors capable of expressing in said cells a nucleoprotein and an RNA-dependent RNA polymerase; and (c) culturing said cells such that RNPs are formed and the recombinant negative-strand RNA virus is produced in the absence of helper virus. In accordance with the present invention, the expression vector may be engineered to express the genomic RNA segments, the nucleoprotein and the RNA-dependent polynerase, or any combination thereof. In another embodiment, each component may be provided to the cell in individual expression vectors.
In another yet another embodiment, infectious recombinant negative-strand RNA viruses are rescued in 293T cells by a method comprising introducing expression vectors capable of expressing in said cells genomic RNAs or antigenomic RNAs in cells which express a nucleoprotein and an RNA dependent polymerase and culturing said cells such that RNP""s are formed and the virus is produced in the absence of helper virus.
The present invention also provides methods of generating an infectious recombinant negative-strand RNA viruses having greater than 3 genomic vRNA segments in host cells, said methods comprising: (a) expressing genomic vRNA segments or the corresponding cRNAs from a first set of expression vectors in said cells; and (b) expressing a nucleoprotein and an RNA-dependent RNA polymerase from a second set of recombinant expression vectors in said cells, whereby ribonucleoprotein complexes are formed and the infectious recombinant negative-strand RNA viruses are produced in the absence of helper virs. Preferably, the infectious recombinant negative-strand RNA virus generated is a member of the Orthomyxoviridae family and most preferably the infectious recombinant negative-strand RNA virus generated is an influenza virus.
In one embodiment, an infectious recombinant negative-strand RNA virus having greater than 3 genomic vRNA segments is generated in host cells by a method comprising: (a) introducing a first set of expression vectors capable of expressing in said cells genomic vRNA segments or the corresponding cRNAs; (b) introducing a second set of expression vectors capable of expressing in said cells a nucleoprotein and an RNA-dependent RNA polymerase; and (c) culturing said cells such that RNPs are formed and the infectious recombinant negative-strand RNA virus is produced in the absence of helper virus.
In another embodiment, an infectious recombinant negative-strand RNA virus having greater than 3 genomic vRNA segments is generated in a host cell line expressing a nucleoprotein and an RNA-dependent RNA polymerase by a method comprising: (a) introducing expression vectors capable of expressing in said cell line genomic vRNA segments or the corresponding cRNAs; and (b) culturing said cells such that RNPs are formed and the infectious recombinant negative-strand RNA virus is produced in the absence helper virus. In another embodiment, an infectious recombinant negative-strand RNA virus having greater than 3 genomic vRNA segments is generated in a mammalian cell line expressing genomic vRNA segments or the corresponding cRNAs by a method comprising: (a) introducing expression vectors capable of expressing a nucleoprotein and an RNA-dependent RNA polymerase; and (b) culturing said cells such that RNPs are formed and the infectious recombinant negative-strand RNA virus is produced in the absence of helper virus.
The present invention is based, in part, on Applicants"" identification of the correct nucleotide sequence of the 5xe2x80x2 and 3xe2x80x2 termini of the negative-sense genomes RNA of NDV. The nucleotide sequence of the 3xe2x80x2 termini of the NDV negative-sense genome RNA of the present invention differs significantly from the NDV 3xe2x80x2 termini sequence previously disclosed by Collins et al. in Fundamental Virology 3rd Ed. 1996 by Lippincott-Raven Publishers as shown in FIG. 6. The identification of the correct nucleotide sequence of the NDV 3xe2x80x2 termini allows for the first time the engineering of recombinant NDV RNA templates, the expression of the recombinant RNA templates and the rescue of recombinant NDV particles. Accordingly, the present invention provides methods of generating an infectious, replicating recombinant Newcastle disease virus NV) in mammalian cells, said methods comprising: (a) expressing a genomic vRNA or the corresponding cRNA from an expression vector in said cells; and (b) expressing a nucleoprotein and an RNA-dependent RNA polynerase from a set of expression vectors in said cells, whereby ribonucleoprotein complexes are formed and the recombinant NDV is produced in the absence of helper virus.
In one embodiment, an infectious recombinant NDV is generated in host cells by a method comprising: (a) introducing an expression vector capable of expressing in said cells igenomic vRNA or the corresponding cRNA; (b) introducing a set of expression vectors capable of expressing in said cells a nucleoprotein and an RNA-dependent RNA polymerase; and (c) culturing said cells such that RNPs are formed and recombinant NDV is produced in the absence of helper virus.
In another embodiment, an infectious recombinant NDV is generated in a host cell line expressing a nucleoprotein and an RNA-dependent RNA polymerase by a method comprising: (a) introducing expression vectors capable of expressing in said cell line a genomic vRNA or the corresponding cRNA; and (b) culturing said cell line such that RNPs are formed and recombinant NDV is produced in the absence helper virus. In another embodiment, an infectious recombinant NDV is generated in a host cell line expressing a genomic vRNA segment or the corresponding cRNA by a method comprising: (a) introducing expression vectors capable of expressing in said cell line a nucleoprotein and an RNA-dependent RNA polymerase; and (b) culturing said cell line such that RNPs are formed and recombinant NDV is produced in the absence of helper virus.
The ability to reconstitute negative-strand RNA viruses intracellularly allows the design of novel recombinant viruses (i.e., chimeric viruses) which express heterologous nucleic acid sequences or which express mutant viral genes. The heterologous sequences may encode, for example, epitopes or antigens of pathogens or tumors. The ability to reconstitute negative-strand RNA viruses intracellularly also allows the design of novel recombinant viruses (i.e., chimeric viruses) which express genes from different strains of viruses. Thus, the present invention provides methods of generating chimeric viruses which express heterologous nucleic acid sequences, mutant viral genes, or viral genes from different strains of virus intracellularly from expression vectors.
The present invention provides for the use of the recombinant negative-strand RNA viruses or chimeric viruses of the invention to formulate vaccines against a broad range of viruses and/or antigens including tumor antigens. The recombinant negative-strand RNA viruses or chimeric viruses of the present invention may be used to modulate a subject""s immune system by stimulating a humoral immune response, a cellular immune response or by stimulating tolerance to an antigen. When delivering, tumor antigens, the invention may be used to treat subjects having a disease amenable to immunity mediated rejection, such as non-solid tumors or solid tumors of small size. It is also contemplated that delivery of tumor antigens by the recombinant negative-strand RNA viruses or chimeric viruses described herein will be useful for treatment subsequent to removal of large solid tumors. The recombinant negative-strand RNA viruses or chimeric viruses of the invention may also be used to treat subjects who are suspected of having cancer.
The present invention also provides for the use of the recombinant negative-strand RNA viruses or chimeric viruses of the invention in pharmaceutical compositions for the administration of peptides or polypeptides to a subject.
As used herein, the following terms will have the meanings indicated:
cRNA=anti-genomic RNA
HIV=human immunodefiency virus
L=large protein
M=matrix protein (lines inside of envelope)
MDCK=Madin Darby canine kidney cells
MDBK=Madin Darby bovine kidney cells
MLP=adenovirus type 2 major late promoter linked to a synthetic sequence comprising the spliced tripartite leader sequence of humanadenovirus type 2
moi=multiplicity of infection
NA=neuraminidase (envelope glycoprotein)
NDV=Newcastle disease Virus
NP=nucleoprotein (associated with RNA and required for polymerase activity)
NS=nonstructural protein (function unknown)
nt=nucleotide
PA, PB1, PB2=RNA-directed RNA polymerase components
pA=polyadenylation sequence from SV40
POLI=truncated human RNA polymerase I promoter
R=genomic hepatitis virus ribozyme
RNP=ribonucleoprotein (RNA, PB2, PB1, PA and NP)
rRNP=recombinant RNP
vRNA=virus RNA
viral polymerase complex=PA, PB1, PB2 and NP
WSN=influenza A/WSN/33 virus
WSN-HK virus=reassortment virus containing seven genes from WSN virus and the NA gene from influenza A/HK/8/68 virus
The term xe2x80x9cexpression vectorsxe2x80x9d as used herein refers to plasmids, viral vectors, recombinant nucleic acids and cDNA. Preferably, the term xe2x80x9cexpression vectorsxe2x80x9d refers to plasmids.
The term xe2x80x9chelper virusxe2x80x9d as used herein refers to a virus homologous to the virus being rescued. The helper virus generally supplies one or more of the viral proteins which are required for the production of infectious recombinant negative-strand RNA viruses.
FIG. 1. Schematic representation of a method of generating recombinant influenza virus. Eight transcription plasmids encoding the vRNA segments of an influenza A virus and four protein expression plasmids encoding influenza A virus nucleoprotein and RNA-dependent RNA polymerase subunits are cotransfected into cultured Vero cells (African green monkey kidney cells). Then, MDBK (Madin-Darby bovine kidney) cells are employed for plaque assay and amplification of rescued viral particles.
FIG. 2. Schematic representation of the NDV minigenome. Top illustration depicts the PNDVCAT plasmid including the T7 promoter; the 5xe2x80x2 terminal sequence (5xe2x80x2 end of genomic RNA, 191nt); the inserted nucleotides (CTTAA); 667nt of CAT ORF; the 3xe2x88x9d terminal sequence (3xe2x80x2 end of genomic RNA, 121 nt) the BbS1 and nuclease sites. Lower illustration depicts the chimeric NDV-CAT RNA resulting from in vitro transcription.
FIGS. 3A-3C. Schematic representation of the PTMI expression vectors.
PTM1-NP encodes the NDV NP protein.
PTM1-P encodes the NDV P protein.
PTM1-L encodes the NDV L protein.
FIG. 4. RNA sequence of NDV 5xe2x80x2 and 3xe2x80x2 non-coding terminal regions (plus-sense). Sequences 5xe2x80x2 to the CAT gene represent 121nt of the 5xe2x80x2 non-coding terminal region of NDV plus sense genome comprising 65nt of the leader sequence (in bold) followed by 56nt of the NP gene UTR (SEQ ID NO: 1). Sequences 3xe2x80x2 to the CAT gene represent inserted nucleotides cuuaa (in lower case (positions 1-5 of SEQ ID NO: 2)) and 191nt of the non-coding terminal region of NDV plus sense genome comprising 127nt of the UTR of the L gene followed by 64nt of the trailer region (in bold (positions 6-196 of SEQ ID NO: 2)).
FIGS. 5A-5B. Schematic representation of a structure of recombinant NDV clones. FIG. 4B, representation of infectious NDV expressing HIV Env and Gag. Top panel, HIV Env and Gag are between the M and L genes. Lower panel, HIV Env and Gag are 3xe2x80x2 to the NP gene.
FIG. 6. Schematic representation of the 3xe2x80x2 termini of NDV (SEQ ID NO: 3) as aligned with sequence of Collins et al. Parainfluenza viruses, in Field""s Virology, 3rd ed. B. N. Fields, D. M. Knipe, p.m. Howley et al, eds., Lippincott-Raven Publishing, Philadelphia, 1996 (SEQ ID NO: 4).